Stable growth hormone compounds resistant to proteolytic degradation

ABSTRACT

The present invention relates to growth hormone (GH) compounds having additional disulphide bridges and at least one additional single point mutation making the compounds resistant to proteolytic degradation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371national stage application of International Patent Application PCT/EP2011/050909 (published as WO 2011/089250), filed Jan. 24, 2011, which claimed priority of European Patent Application 10151405.7, filed Jan. 22, 2010, and International Patent Application PCT/CN2010/001120, filed Jul. 23, 2010; this application further claims priority under 35 U.S.C. §119 of U.S. Provisional Applications 61/297,305, filed Jan. 22, 2010, and 61/378,602, Aug. 31, 2010.

FIELD OF INVENTION

The present invention relates to stable growth hormone (GH) compounds resistant to proteolytic degradation.

INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING

In accordance with 37C.F.R. §1.52(e)(5), Applicants enclose herewith the Sequence Listing for the above-captioned application entitled “SEQUENCE LISTING”, created on Jan. 24, 2011. The Sequence Listing is made up of 1.95 kilobytes, and the information contained in the attached “SEQUENCE LISTING” is identical to the information in the specification as originally filed. No new matter is added.

BACKGROUND OF INVENTION

Growth hormone (GH) is a polypeptide hormone secreted by the anterior pituitary in mammals. Dependent on species GH is a protein composed of approximately 190 amino acid residues corresponding to a molecular weight of approximately 22 kDa. GH binds to and signals through cell surface receptors, the GH receptors (GHR). GH plays a key role in promoting growth, maintaining normal body composition, anabolism and lipid metabolism. It also has direct effects on intermediate metabolism, such as decreased glucose uptake, increased lipolysis, increased amino acid uptake and protein synthesis. The hormone also exerts effects on other tissues including adipose tissue, liver, intestine, kidney, skeleton, connective tissue and muscle. Recombinant hGH has been produced and commercially available as, for ex: Genotropin™ (Pharmacia Upjohn), Nutropin™ and Protropin™ (Genentech), Humatrope™ (Eli Lilly), Serostim™ (Serono), Norditropin (Novo Nordisk), Omnitrope (Sandoz), Nutropin Depot (Genentech and Alkermes). Additionally, an analogue with an additional methionine residue at the N-terminal end is also marketed as, for ex: Somatonorm™ (Pharmacia Upjohn/Pfizer).

GH shares a common topology with the other members of the GH-family of proteins, Prolactin (PRL) and Placental Lactogen (PL). GH is classified as a four-helix bundle protein (FIG. 1) exhibiting an “up-up-down-down” topology with two conserved disulphide linkages. Specifically, wild-type human GH (hGH) is composed of 191 amino acid residues and has four cysteine residues at positions 53, 165, 182 and 189, which stabilizes the three dimensional structure of the protein by forming two intramolecular disulphide bonds connecting C53 with C165 and C182 with C189, respectively (FIG. 1). The structure of hGH has been experimentally determined by X-ray crystallography in the free form (Chantalet L. et al (1995) Protein and Peptide Letters 3, 333-340) and in complex with its binding protein (the extra cellular domain of the human GHR (hGHR)) (Devos, A. M. et al (1992) Science 255, 306-312). These structures have been deposited in the Protein Data Bank (PDB) and are publicly available (PDB accession codes 1HGU and 1HWG, respectively). Thus, from the published hGH structures residues important for hGH binding to hGHR can be identified. Furthermore, the dynamic properties of hGH has been studied by Nuclear Magnetic Resonance (NMR) spectroscopy (Kasimova M. R. et al. J. Mol. Biol.(2002) 318, 679-695). In combination, the X-ray and NMR data can distinguish regions of hGH which are well structured and well defined from regions which are less structured and dynamic. Less structured and dynamic regions of hGH are expected to be particularly susceptible to proteolytic cleavage and proper stabilization of such regions would lead to improved proteolytic stability.

hGH has been subject to extensive mutagenesis in attempts to produce hGH analogues with desired chemical or biological properties. Specifically, cysteine mutants for several purposes have been described.

US 2003/0162949 disclose cysteine variants of members of the GH supergene family. A general method is provided for creating site-specific, biologically active conjugates of these proteins. The method involves adding cysteine residues to non-essential regions of the proteins or substituting cysteine residues for non-essential amino acids in the proteins using site-directed mutagenesis and then covalently coupling a cysteine-reactive polymer or other type of cysteine-reactive moiety to the proteins via the added cysteine residue

WO 02/055532 describes genetically engineered hGH mutants having at least one non-polypeptide moiety covalently attached, particularly hGH mutants where a introduced cysteine residue was used for pegylation.

U.S. Pat. No. 5,951,972 describes physiologically active derivatized natural and recombinant mammalian and human proteins and polypeptides wherein at least one-naturally-occurring or incorporated cysteine residue within the protein is derivatized with various substituents.

The proteolytic cleavage of hGH has been studied in detail. The long loop composed of residues 128 to 154 has putative cleavage sites for several proteases, such as thrombin, plasmin, collagenase, subtilisin and chymotrypsin-like serine proteases. Accordingly, this part of hGH has been shown to be particularly susceptible to proteolytic cleavage (Lewis, U. J. Ann. Rev. Physiol. (1984.) 46, 33-42). Enzymes reported to degrade hGH include thrombin, plasmin, subtilisin, chymotrypsin-like serine proteinases and kallikreins.

The degradation of hGH in rat tissue has been investigated (Garcia-Barros et al. J. Endocrinol. Invest. (2000) 23, 748-754).

In rat thyroid gland chymotrypsin-like proteases, favouring cleavage at bulky and lipophilic amino acid residues, were found initially to cleave the peptide bond between Y143 and S144 resulting in a two chain molecule, followed by cleavage between Y42 and S43, liberating the N-terminal peptide F1-Y42. The split loop in the two chain molecule is processed further by cleavage between F146 and D147 by chymotrypsin-like proteases and further by the action of carboxypeptidases.

Several methods to produce hGH analogues stabilized towards proteolytic degradation have been reported.

Alam et al., J. Biotech. 65, 183-190 (1998)) designed hGH mutants resistant to thrombin and plasmin by specific point mutations. Thrombin cleaves hGH specifically between R134 and T135, and the double mutant R134D, T135P yielded a hGH variant resistant to cleavage by thrombin, and the triple mutant R134D, T135P, K140A resulted in resistance to plasmin. Furthermore, the latter hGH mutant was resistant to proteolysis by human plasma over a period of 7 days.

EP534568 describes hGH mutants stabilized towards proteolytic degradation by mutating R134 to alanine, leucine, threonine, phenylalanine, proline or histidine.

WO2004022593/Nautilus describes general high through-put directed evolution methods to produce modified cytokines, including GH variants, with increased proteolytic stability.

WO2006048777/Nautilus specifically describes modified hGH analogues with improved proteolytic stability. The analogues contain one to five mutations at positions 1-55, 57, 58, 60-63, 67-87, 89-91, 93, 95-100, 102-128, 131-132, 135-139, 141, 142, 144, 148-182, 184, 185 and 187-191. Introduction of cysteine residues can potentially lead to the formation of undesired disulfide linked dimers and in WO2006048777 the substitution of amino acid residues by cysteine is specifically excluded from the scope; in WO2006048777 (p. 65) it is stated: “The replacement of amino acids by cysteine residues is explicitly avoided since this change would potentially lead to the formation of intermolecular disulfide bonds”.

There is an obvious need to develop hGH compounds which are resistant to proteolytic degradation. Such stabilized compounds should exhibit increased stability towards proteolytic cleavage while retaining the desired biological properties of hGH. Such GH molecules would have increased stability, slower clearance and/or prolong in vivo half-life.

Furthermore protein therapeutics generally needs to be administered intravenously or subcutaneously because they are generally not sufficiently orally available. The low oral bioavailability of proteins is partly due to proteolytic degradation in the gastrointestinal tract. Hence, there is also a need to develop hGH compounds that can be administered orally to treat hGH related disorders.

SUMMARY OF INVENTION

The present invention relates to growth hormone compounds comprising one or more additional disulfide bond(s) and at least one additional single point mutation compared to human growth hormone. In the GH compounds of the present invention at least one additional cysteine residue has been introduced by mutating at least one amino acids in the wild-type hGH sequence to cysteine. In the GH compounds of the present invention the site(s) of mutation are chosen in such a way that (1) the introduced cysteine residue(s) is/are appropriately placed in the three dimensional structure of the folded protein to allow for the formation of one or more additional disulphide bonds not present in the wild type protein (2) the native structure of hGH is not disrupted (3) the GH compound exhibits increased stability towards proteolytic cleavage compared to wild type hGH or other enhanced functionalities and (4) the GH compound retains the desired biological activities associated with wild type hGH. Such disulphide variants of hGH being resistant to proteolytic degradation in the gastro intestinal tract can be developed as orally administered drugs for treating hGH related disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Structure of hGH bound to two copies of the hGH binding protein (PDB 1HWG). The four major helices in hGH are shown in dark gray and are labelled H1-H4. The directions (N→C terminal) are indicated by arrows. The N- and C-termini of hGH are labelled N and C, respectively. The two disulphide bonds connecting C53 with C165 and C182 with C189, respectively, are represented by black sticks and balls. Also labeled are L128 and D154 representing the first and last residues, respectively, in the long flexible loop connecting H3 and H4.

FIG. 2 Wild type amino acid sequence of hGH with the four main helices (H1-H4) highlighted and labelled. Also labelled are the three loops (L1-L3) connecting the main helices. The helix definitions refer to hGH in complex with its binding protein (PDB 1HWG).

DEFINITIONS

In the present context, the words “human growth hormone (hGH)’, “hGH wt” and “wild type hGH (wthGH)” are used interchangeably and refer both to a protein with an amino acid sequence as SEQ ID No. 1.

In the present context, the terms “peptide” and “polypeptide” are used interchangeably and are intended to indicate the same. The terms “peptide” or “polypeptide” are intended to indicate a sequence of two or more amino acids joined by peptide bonds, wherein said amino acids may be natural or unnatural. The constituent amino acids may be from the group of the amino acids encoded by the genetic code and they may be natural amino acids which are not encoded by the genetic code, as well as synthetic amino acids. Natural amino acids which are not encoded by the genetic code are e.g. Hyp (hydroxyproline), y-carboxyglutamate, Orn (ornithine), phosphoserine, D-alanine and D-glutamine. Synthetic amino acids comprise amino acids manufactured by chemical synthesis, such as D-isomers of the amino acids encoded by the genetic code such as D-alanine and D-leucine, Aad (α-aminoadipic acid), Aib (α-aminoisobutyric acid), Abu (α-aminobutyric acid), Agl (α-amino-glycine), Asu (α-aminosuberic acid), Cha (β-cyclopentyl-alanine), Chg (cyclohexyl glycine), Dab (α,γ-diaminobutyric acid), Dap (α, β-diaminopropanic acid), Hcy (homocysteine), Hpr (homoproline), Nle (Norleucine), Phg (phenylglycine), Hph (homophenylalanine), 1Nal (β-(1-naphthyl-alanine), 2Nal (β-(2-naphthyl-alanine), 2 Pal (-(2-pyridyl)-alanine, 3 Pal (β-(3-pyridyl)-alanine), Pip (4-amino-piperidine-4-carboxylic acid), Pra (propargyl-glycine), Pyr (pyroglutamic acid), Gla (γ-carboxy-glutamic acid), Hci (homocitruline), Nva (norvaline), Tle (tert-butylglycine), β-alanine, 3-aminomethyl benzoic acid and anthranilic acid.

The term also encompasses the term “proteins”, which may consists of one polypeptide chain, or two or more polypeptide chains held together by non-covalent or covalent interactions, such as for instance cysteine bridges.

It is to be understood that the term is also intended to include peptides, which have been derivatized, for instance by attaching moieties such as, but not limited to, PEG, carbohydrates, fatty acids, albumin binders, alkyl chains, lipophilic groups, vitamins, bile acids, or spacers to the side chains or main chain of the peptide in addition to comprising the additional disulfide bonds. The term peptide includes any suitable peptide and may be used synonymously with the terms polypeptide and protein, unless otherwise stated or contradicted by context, provided that the reader recognize that each type of respective amino acid polymer-containing molecule may be associated with significant differences and thereby form individual embodiments of the present invention (for example, a peptide such as an antibody, which is composed of multiple polypeptide chains, is significantly different from, for example, a single chain antibody, a peptide immunoadhesin, or single chain immunogenic peptide). Therefore, the term peptide herein should generally be understood as referring to any suitable peptide of any suitable size and composition (with respect to the number of amino acids and number of associated chains in a protein molecule). Moreover, peptides described herein may comprise non-naturally occurring and/or non-L amino acid residues, unless otherwise stated or contradicted by context.

The term peptide, unless otherwise stated or contradicted by context, (and if discussed as individual embodiments of the term(s) polypeptide and/or protein) also encompasses derivatized peptide molecules. A derivatized peptide molecules is one in which one or more of the amino acid residues of the peptide have been chemically modified (for instance by alkylation, acylation, ester formation, or amide formation) or associated with one or more non-amino acid organic and/or inorganic atomic or molecular substituents (for instance a polyethylene glycol (PEG) group, a lipophilic substituent (which optionally may be linked to the amino acid sequence of the peptide by a spacer residue or group such as β-alanine, γ-aminobutyric acid (GABA), L/D-glutamic acid, succinic acid, and the like), a fluorophore, biotin, a radionuclide, etc.) and may also or alternatively comprise non-essential, non-naturally occurring, and/or non-L amino acid residues, unless otherwise stated or contradicted by context (however, it should again be recognized that such derivatives may, in and of themselves, be considered independent features of the present invention and inclusion of such molecules within the meaning of peptide is done for the sake of convenience in describing the present invention rather than to imply any sort of equivalence between naked peptides and such derivatives).

Non-limiting examples of such amino acid residues include for instance 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, β-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline, norvaline, norleucine, ornithine, propargyl-glycine and statine halogenated amino acids.

A “compound” described in the present invention may be a “protein” or “peptide” or “polypeptide” which may be an “analogue” or a “derivative” or a “variant”, which retains desired biological activities similar to wthGH, irrespective to the manner it has been modified.

The term “analogue” or “variant” as used herein when referring to a polypeptide, means a modified version of said peptide wherein one or more amino acid residues of the peptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the peptide and or wherein one or more amino acid residues have been added to the peptide. Such substitution or addition or deletion of amino acid residues can take place at the N-terminal of the peptide and/or at the C-terminal of the peptide and/or in between N- or C-terminal of the peptide. All amino acids for which the optical isomer is not stated are to be understood to mean the L-isomer.

The terms “disulphide bond” or “disulphide bridge” are used interchangeably and intended to indicate the same. A “disulphide bond” or “disulphide bridge” in proteins is formed between the thiol groups of cysteine residues.

The term “additional cysteine” or “introduced cysteine” are used interchangeably and are intended to indicate the same. The terms are intended to include a cysteine residue not present in wild type hGH. To minimize structural changes the cysteine residue(s) are usually introduces by substitution of amino acid residue(s), whereby the length of hGH is maintained. Insertion of an additional cys residue may be tolerated in loop sections or at the boarders of the helixes, whereas introduction of cys residues within the helix'es is less attractive.

The term “additional disulphide bond” or “introduced disulphide bond” are used interchangeably and are intended to indicate the same. The terms are intended to include disulphide bonds formed between two cysteine residues of which at least one is not present in wild type hGH.

The term “additional single point mutation” is used herein to indicate a mutation (compared to hGH as defined in SEQ ID NO 1), which is unrelated to the disulphide bridge forming additional cysteines introduced into the growth hormone compound. The term “additional single cys mutation” may be used to specify a point mutation which introduces a cysteine residue. Such a mutation may be the “additional single point mutation” or an even further mutation in hGH.

The term “derivative” as used herein refers to a peptide or polypeptide, wherein one or more amino acid residues of the peptide have been chemically modified by introduction of a polymer such as PEG, carbohydrate moieties, albumin binders, fatty acids, lipophilic groups, vitamins, bile acids or spacers to the side chains or main chain of the growth hormone compound. The chemical modifications may also be transient in nature, i.e. they may readily be removed in vivo. The chemical modifications can be post-translationally introduced, for instance by the cell itself or by chemical modifications performed on the peptide after expression.

The term “identity” as known in the art, refers to a relationship between the sequences of two or more peptides, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between peptides, as determined by the number of matches between strings of two or more amino acid residues. “Identity” measures the percent of identical matches between two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”). Identity of related peptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are described in publicly available computer programs. Preferred computer program methods to determine identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well known Smith Waterman algorithm may also be used to determine identity.

For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, Wis.), two peptides for which the percent sequence identity is to be determined are aligned for optimal matching of their respective amino acids (the “matched span”, as determined by the algorithm). A gap opening penalty (which is calculated as 3.times. the average diagonal; the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. A standard comparison matrix (see Dayhoff et al., Atlas of Protein Sequence and Structure, 5, (1978) for the PAM 250 comparison matrix; Henikoff et al., PNAS USA 89, 10915-10919 (1992) for the BLOSUM 62 comparison matrix) is also used by the algorithm.

Preferred parameters for a peptide sequence comparison include the following:

Algorithm: Needleman et al., J. Mol. Biol. 48, 443-453 (1970); Comparison matrix: BLOSUM 62 from Henikoff et al., PNAS USA 89, 10915-10919 (1992); Gap Penalty: 12, Gap Length Penalty: 4, Threshold of Similarity:

The GAP program is useful with the above parameters. The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps) using the GAP algorithm.

The terms “protease or proteases” is intended to include all enzymes possessing the ability to catalyze hydrolytic cleavage of a peptide bond. Proteases may be intra cellular, extra cellular or membrane bound proteases, proteinases or peptidases, and include proteases in the lumen of mammalian intestine and proteases present in mammalian plasma. Proteases may both be of the type endo proteases and exo proteases.

The terms “resistant to proteolytic degradation” or “increased stability towards proteolytic degradation” or “increased stability towards proteolytic cleavage” or “improved proteolytic stability” or “proteolytic stability” are used interchangeably and intended to indicate the same. Used in connection to a hGH compound of the invention, the terms are intended to indicate that the polypeptide chain of said hGH compound is cleaved at a slower rate, compared to wild type hGH, by a protease under specific conditions.

The rate of proteolytic cleavage of a protein may be measure by several techniques known to the person skilled in the art. An example of an assay measuring the rate of degradation of hGH or a hGH compound is described in Example 5.

The invention also relates to methods useful for improving the pharmacological properties of hGH compounds. These pharmacological properties could for instance be an increase in the functional in vivo half-life, the plasma in vivo half-life, the mean residence time, a decrease in the renal clearance.

The term “functional in vivo half-life” is used in its normal meaning, i.e., the time at which 50% of the biological activity of the peptide, for instance a growth hormone compound, wherein the growth hormone compound is still present in the body/target organ, or the time at which the activity of the peptide, for instance growth hormone compound is 50% of its initial value. As an alternative to determining functional in vivo half-life, “in vivo plasma half-life”, and protracted action may be determined, i.e., the time at which 50% of the peptide circulate in the bloodstream prior to being cleared. Determination of plasma half-life is often more simple than determining functional half-life and the magnitude of plasma half-life is usually a good indication of the magnitude of functional in vivo half-life.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to GH compounds having one or more additional disulphide bond(s) and at least one additional single point mutation compared to hGH. The disulphide bonds are formed between pairs of cysteines of which one or both are introduced by point mutations in the wild type hGH sequence. The sites of mutation are chosen such that the introduced cysteine residues are appropriately placed in the three dimensional structure of the folded protein to allow for the formation of a disulphide bond. The folded protein with the additional disulphide bond may be obtained by expressing the appropriate cysteine mutant of hGH in soluble form by a suitable host organism, or recovered from inclusion bodies using standard refolding conditions for growth hormone compounds, which are well known to those skilled in the art (Cabrita and Bottomley, Biotechnology Annual Review 10, 31-50 (2004)). The identification of candidate positions for introduction of additional disulphide bonds can be aided by computational methods, e.g. using the experimentally determined three dimensional structure of hGH (PDB accession code 1HWG) in complex with two copies of its binding protein. Selection of appropriate positions for introduction of disulphide bond can be based distance and geometry criteria for disulphide bonds described in Dombkowski A., A., Bioinformatics 19, 1852-1853 (2003) and Petersen et al., Protein Eng. 12, 535-548 (1999).

The cysteine mutants are chosen such that the introduced disulphide bonds do not disrupt the native structure of the protein and have minimal negative impact on the desired biological activity associated with hGH. Thus, the compounds are constructed such that the introduced disulphide bonds do not impair interaction with hGHR. The regions in hGH important for receptor interaction have been identified from 1HWG. Thus, the selection of appropriate positions for introducing disulphide bonds, which are neutral with respect to biological activity, can be guided by analyzing the 1HWG structure.

The cysteine mutants may be chosen such that the introduced disulphide bonds provide increased stability towards proteolytic cleavage. The susceptibility of a protein to protease cleavage is defined in part by the primary amino acid sequence of said protein. Proteases may be relatively unspecific or may, with variable degree of selectivity, recognize specific motifs in the primary amino acid sequence. However, the three dimensional structure and dynamics of the protein molecule acting as a substrate strongly influence proteolytic stability. Highly flexible and dynamic loop structures are particularly vulnerable to protease catalyzed cleavage, whereas well structured regions are generally less so. Thus, protection against proteolytic cleavage can be obtained by stabilizing dynamic regions of a protein by introducing disulphide bonds.

One aspect of the invention relates to a growth hormone compound comprising one or more additional disulfide bond(s) and at least one additional single point mutation compared to hGH as defined by SEQ ID NO 1. As described herein below the polypeptide of a growth hormone compound according to the invention preferably has a high level of identity to human growth hormone identified by SEQ ID NO 1.

Accordingly, one embodiment of present invention provides stable GH compounds made resistant to proteolytic degradation by introduction of one or more additional disulphide bonds in hGH as defined by SEQ ID No. 1. In one embodiment the growth hormone compound is stabilized towards proteolysis which is further described below.

In one embodiment of the present invention the proteolytic stability of growth hormone compound is achieved by introducing a disulphide bond between a loop segment and a helical structure.

In one embodiment of the present invention the proteolytic stability of growth hormone compound is achieved by introducing a disulphide bond within a loop segment.

In one embodiment of the present invention the proteolytic stability of growth hormone compound is achieved by introducing a disulphide bond between loop segments.

In one embodiment of the present invention the proteolytic stability of growth hormone compound is achieved by introducing a disulphide bond between helices.

In one embodiment of the present invention at least one of the introduced disulphide bonds links two cysteine residues of a growth hormone compound, wherein at least one of said cysteine residues is not present in wild type hGH.

In one embodiment of the present invention the introduced disulphide bonds of a growth hormone compound are positioned between cysteine residues that are selected using distance and geometry criteria described in Dombkowski A., A., Bioinformatics 19, 1852-1853 (2003) and Petersen et al., Protein Eng. 12(7), 535-548 (1999).

In one embodiment of the present invention the introduced disulphide bond(s) of the growth hormone compound stabilize the loop connecting H3 and H4 (L3, residues 128-154), i.e. at least one of the cysteines in the introduced disulphide bond is positioned in the segment comprising residues 128-154 (FIGS. 1 and 2).

As described above the invention relates to a growth hormone compound comprising an additional disulfide bond connecting a loop segment and a helical segment or within a loop segment or connecting loop segments or connecting helical segments of the polypeptide. The location of any such additional disulfide bond is for the purpose of this application described with reference to the polypeptide of hGH as defined in SEQ ID NO 1. Non limiting examples of cys mutations providing linkage connecting helix's, loops and a loop and a helix are listed in table 1 below.

TABLE 1 First Amino Acid as Second Amino Acid Secondary defined by sequence as defined by Structural alignment with sequence alignment segments SEQ ID NO 1. with SEQ ID NO 1. connected^(a). 1. 16 117 H1-H3 2. 17 174 H1-H4 3. 21 170 H1-H4 4. 26 102 H1-L2 5. 26 103 H1-L2 6. 47 50 L1-L1 7. 49 161 L1-L1 8. 54 143 L1-L3 9. 54 144 L1-L3 10. 54 146 L1-L3 11. 55 143 L1-L3 12. 57 143 L1-L3 13. 58 141 L1-L3 14. 58 143 L1-L3 15. 58 144 L1-L3 16. 59 137 L1-L3 17. 61 66 L1-L1 18. 61 67 L1-L1 19. 71 132 L1-L3 20. 73 132 H2-L3 21. 73 139 H2-L3 22. 77 138 H2-L3 23. 77 139 H2-L3 24. 81 141 H2-L3 25. 81 143 H2-L3 26. 84 143 H2-L3 27. 84 144 H2-L3 28. 85 143 H2-L3 29. 85 144 H2-L3 30. 89 146 H2-L3 31. 92 146 H2-L3 32. 92 148 H2-L3 33. 94 107 H2-H3 34. 102 105 L2-H3 35. 156 146 H4-L3 36. 156 148 H4-L3 37. 185 188 Ct-Ct ^(a)H1-H4 refer to helix 1-4, L1-L3 refer to loops 1-3, and Ct refer to C-terminal segment.

In one embodiment a growth hormone compound according to the invention comprises at least one pair of mutations corresponding to R16C/L117C, A17C/E174C, H21C/M170C, D26C/V102C, D26C/Y1030, N47C/T50C, Q49C/G161C, F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, P61C/E66C, P61C/T67C, S71C/S132C, L73C/S132C, L73C/F139C, R77C/11380, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C and/or V185C/S188C in SEQ ID NO 1.

In one embodiment a growth hormone compound comprises at least one pair of mutations corresponding to A17C/E1740, H21C/M170C, D26C/V102C, D26C/Y103C, F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C, F92C/T148C and/or R94C/D107C in SEQ ID NO 1.

One embodiment according to the invention relates to a growth hormone compound comprising an additional disulfide bond wherein at least one of the cysteines is present in L3 (AA 128-154 in SEQ ID NO 1), or such as in the middle region of the loop defined by AA 135-148) or corresponding amino acid residues.

In one embodiment a growth hormone compound at least one of the cysteines of the additional disulfide bond is present in L3, in a position corresponding to AA 141, AA142, AA143, AA144, AA145 or AA146, preferably AA143 or AA144 in SEQ ID NO 1.

In one embodiment a growth hormone compound comprises at least one pair of mutations corresponding to F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L73C/S132C, L73C/F139C, R77C/I1380, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C and/or F92C/T148C in SEQ ID NO 1.

In one embodiment a growth hormone compound comprises at least one pair of mutations corresponding to F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C and/or F92C/T148C in SEQ ID NO 1.

One embodiment according to the invention relates to a growth hormone compound comprising an additional disulfide bond connecting L3 with L1.

In one embodiment a growth hormone compound comprises an additional disulfide bond connecting an amino acid residue corresponding to AA54, AA55, AA56, AA57, AA58 or AA59 in L3 with an amino acid corresponding to AA143 or AA144 in L1 of SEQ ID NO 1.

In one embodiment a growth hormone compound according to the invention comprises at least one pair of mutations corresponding to F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C and/or S71C/S132C in SEQ ID NO 1.

One embodiment according to the invention relates to a growth hormone compound comprising an additional disulfide bond connecting L3 with a helical segment, such as helix 2 (H2).

In one embodiment a growth hormone compound comprises an additional disulfide bond connecting an amino acid residue corresponding to AA84 or AA85 in H2 with an amino acid corresponding to AA143 or AA144 in L3 of SEQ ID NO 1.

In one embodiment a growth hormone compound comprises at least one pair of mutations corresponding to L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C and F92C/T148C in SEQ ID No.

In one embodiment a growth hormone compound comprises at least one pair of mutations corresponding to L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C and/or F92C/T148C in SEQ ID NO 1.

One embodiment according to the invention relates to a growth hormone compound comprising an additional disulfide bond connecting L2 with helix 1.

In one embodiment a growth hormone compound comprises at least one pair of mutations corresponding to D26C/V102C or D26C/Y103C.

For disulphide bridges between two cysteine residues, the cysteine residues may be introduced or substituted in any of the regions or positions as defined herein before in order to facilitate formation of one or more additional disulphide bonds. Substitution and insertions of amino acid residues can be carried out by standard techniques known to a person skilled in the art.

One embodiment of the present invention relates to a growth hormone compound, wherein the polypeptide sequence is at least 80%, such as 90% or such as 95% identical to hGH defined by SEQ ID NO 1. In further embodiments the polypeptide is 96%, 97% or 98% identical to hGH defined by SEQ ID NO 1.

The present invention relates to growth hormone compounds comprising one or more additional disulfide bond(s) and at least one additional single point mutation compared to human growth hormone. The at least one additional single point mutation may be included in the growth hormone compound for various reasons associated with the functionality of the growth hormone compound. Said at least one additional single point mutation may be aimed at further increasing protease stability and/or providing a “site” suitable for chemical modification, e.g. by introduction of amino acid residues comprising chemical entities suitable for chemical modification.

The invention thus relates to any growth hormone compound comprising one or more additional disulfide bond(s), as described herein, and at least one additional single point mutation compared to human growth hormone as defined by SEQ ID NO 1.

In one embodiment according to any of the invention the at least one additional single point mutation of the growth hormone compound is at a know protease site.

In one embodiment at least one additional single point mutation is at a position corresponding to position 1-55, 57, 58, 60-63, 67-87, 89-91, 93, 95-100, 101, 102-128, 131-132, 135-139, 141, 142, 144, 148-182, 184, 185 and/or 187-191 of SEQ ID NO 1.

In one embodiment at least one additional single point mutation is at a position corresponding to position 10, 40, 41, 42, 55, 57, 62, 101, 134, 136, 139, 142 and/or 144 of SEQ ID NO 1.

In one embodiment the at least one additional single point mutation is in a position corresponding to position 55, 57 62, 101, 134, 136, 142 and/or 144 of SEQ ID NO 1.

In one embodiment the at least one additional single point mutation is in a position corresponding to position 62, 101, 134, 136, 142 and/or 144 of SEQ ID NO 1.

In one embodiment at least one additional single point mutation is present in L1 (corresponding to AA 36-71 of SEQ ID NO 1) and/or L3 (corresponding to AA 128-154 of SEQ ID NO 1) and in a further embodiment at least one additional single point mutation is present in L1. In one embodiment at least one additional single point mutation is present in the middle region of L1 (corresponding to AA 40-65 of SEQ ID NO 1). In one embodiment at least one additional single point mutation is present in a position corresponding to position 40, 41, 42, 55, 57 and/or 62 of SEQ ID NO 1. In one embodiment at least one additional single point mutation is present in L3 and in a further embodiment at least one additional single point mutation is present in the middle region of L3 (corresponding to AA 134-148 of SEQ ID NO 1).

In one embodiment at least one additional single point mutation is in a position corresponding to position 134, 136, 139, 142 and/or 144 of SEQ ID NO 1.

In one embodiment at least one additional single point mutation is a position corresponding to position 62 of hGH as defined in SEQ ID NO 1. In one such embodiment the Serine (S62) is substituted with an amino acid residue selected from the group of threonine (T), asparagine (N), cysteine (C), histidine (H), glutamine (Q) and glutamic acid (E).

In one embodiment at least one additional single point mutation is a position corresponding to position 55 of hGH as defined in SEQ ID NO 1. In one such embodiment the Serine (S55) is substituted with an amino acid residue selected from the group of threonine (T), asparagine (N), cysteine (C), histidine (H), glutamine (Q) and glutamic acid (E).

In one embodiment at least one additional single point mutation is a position corresponding to position 57 of hGH as defined in SEQ ID NO 1. In one such embodiment the Serine (S57) is substituted with an amino acid residue selected from the group of threonine (T), asparagine (N), cysteine (C), histidine (H), glutamine (Q) and glutamic acid (E).

According to the invention the one or more additional disulfide bond(s) is/are obtained by amino acid substitution of at least two amino acids compared SEQ ID NO 1.

In a further embodiment the compound comprises exactly one additional disulfide bond compared to SEQ ID NO 1.

In one embodiment the polypeptide of a growth hormone compound according to the invention comprises at least two additional cysteines compared to human growth hormone as defined in SEQ ID NO 1.

In a further embodiment the polypeptide comprises exactly two additional cysteines compared to human growth hormone as defined in SEQ ID NO 1.

In one embodiment the polypeptide of a growth hormone compound according to the invention comprises a total of at most 10 amino acid substitutions compared to SEQ ID NO 1. In one embodiment the growth hormone compound comprises a total of at most 8, such as at most 7, such as at most 6, such as at most 5, such as at most 4 amino acid substitutions. In one embodiment the polypeptide of the growth hormone compound according to the invention comprises exactly three amino acid substitutions compared to SEQ ID NO 1.

In one embodiment the growth hormone compound comprises exactly two additional cysteines compared to human growth hormone as defined in SEQ ID NO 1.

In one embodiment the growth hormone compound comprises exactly two additional cysteines and exactly one additional single point mutation compared to human growth hormone as defined in SEQ ID NO 1.

In one embodiment the growth hormone compound comprises exactly three additional cysteines compared to human growth hormone as defined in SEQ ID NO 1.

In one embodiment the growth hormone compound comprises one additional disulphide bond and an additional single cys mutation compared to human growth hormone as defined in SEQ ID NO 1.

In one embodiment the additional single cys mutation is selected from any one of a single cys mutation in the N-terminal, H1, L1, H2, L2 or H3 regions of GH.

In one embodiment the additional single cys mutation is selected from the group of mutation corresponding to: T3C, P5C, S7C, D11C, H18C, Q29C, E300, E33C, A34C, Y35C, S55C, S57C, S62C, E88C, Q91C, S95C, A98C, N99C, S100C, L101C, V102C, Y103C, D107C, S108C, D112C, Q122C and G126C in hGH (SEQ ID NO:1).

In one embodiment the additional single cys mutation is located in a position corresponding to AA 93-106, such as AA 99-106 or AA 99-103 in hGH.

In one embodiment the additional single cys mutation is N99C, S100C or L101C.

In one embodiment the additional single cys mutation is located in a position corresponding to AA 55-62 in hGH.

In one embodiment the additional single cys mutation is S55C, S57C or S62C.

In one embodiment of the present invention the growth hormone compound is chemically modified via attaching moieties such as, but not limited to, PEGs, carbohydrates, albumin binders, fatty acids, alkyl chains, lipophilic groups, vitamins, bile acids, or spacers to the side chains or main chain of the growth hormone compound in addition to comprising additional disulfide bonds.

In one embodiment of the present the growth hormone compound is chemically modified in order to facilitate transport across the epithelia when compared to hGH.

In one embodiment of the present invention a growth hormone compound of the present invention is chemically modified in order to obtain a prolonged duration of in vivo action.

In one embodiment of present invention a growth hormone compound is chemically modified in order to obtain a prolonged duration of functional in vivo half-life.

In one embodiment of present invention the chemical modifications of growth hormone compound may also be transient in nature, i.e. they may readily be removed in vivo.

In one embodiment of the present invention the growth hormone compound modifications can take place at any amino acid residue not interfering with binding of the growth hormone compound to the hGHR.

In one embodiment the growth hormone compound has increased stability towards proteolytic degradation.

In one embodiment of the present invention a growth hormone compound has increased stability towards proteolytic degradation by proteases present in mammalian plasma.

In one embodiment of the present invention a growth hormone compound has increased stability towards proteolytic degradation by a pancreatic protease.

In one embodiment of the present invention a growth hormone compound has increased stability towards proteolytic degradation by proteases present in the gastrointestinal tract. In one embodiment of the present invention a growth hormone compound has increased stability towards proteolytic cleavage.

One embodiment of the present invention relates to a growth hormone compound comprising one or more additional disulfide bond(s) which is stabilized towards degradation by protease(s), such as digestive proteases, such as pepsin, trypsin, chymotrypsin, carboxypeptidase and/or elastases.

In one embodiment of the present invention a growth hormone compound has increased stability towards proteolytic degradation by trypsin, chymotrypsin and/or elastase.

In one embodiment a growth hormone compound is stabilized towards degradation by chymotrypsin and/or elastase.

In one embodiment a growth hormone compound comprises at least one pair of mutations corresponding to H21C/M170, D26C/V102C, D26C/Y103C, F54C/Y143C, F54C/S144C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C and/or S85C/S144C in SEQ ID NO 1.

In one embodiment a growth hormone compound comprises at least one pair of mutations corresponding to D26C/V102C, D26C/Y103C, S57C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, Q84C/Y143C, S85C/Y143C, S85C/S144C, F92C/T148C and/or R94C/D107C in SEQ ID NO 1.

In one embodiment a growth hormone compound comprises at least one pair of mutations corresponding to S57C/Y143C, Q84C/Y143C, S85C/Y143C and/or S85C/S144C in SEQ ID NO 1.

In one embodiment of the present invention a growth hormone compound has increased in vivo half life.

In one embodiment of the present invention a growth hormone compound has increased shelf life.

In one embodiment of the present invention a growth hormone compound may be a fusion protein.

In one embodiment of the present invention a growth hormone compound is a polypeptide comprising an amino acid sequence having at least 20%, such as at least 30%, for instance at least 40%, such as at least 50%, for instance at least 60%, such as at least 70%, for instance at least 80%, such as at least 90% identity, for instance at least 95%, such as at least 96%, for instance at least 97%, such as at least 98% identity to SEQ ID No. 1 and which polypeptide has an activity in the assay described in Example 3 and Example 3A (hyposectomized rats) of at least 1%, such as at least 5%, for instance at least 10%, such as at least 25% of the activity of hGH. To avoid doubt, a growth hormone compound of the invention may also have a higher activity than hGH in these assays. If the growth hormone compound is derivatized in some way, the activity of the growth hormone in relation to hGH should be measured on the underivatized growth hormone compound, as the derivatization may change the activity significantly. For instance in the case of a growth hormone compound derivatized with a property-modifying group that prolongs the functional in vivo half-life of the growth hormone compound, the activity of the derivatized growth hormone compound may be much lower than the activity of hGH, which decrease is counteracting by the prolonged residence time. In one embodiment, the growth hormone compound is a fragment of such a polypeptide, which fragment has retained a significant amount of the growth hormone activity as described above.

In one embodiment of the present invention a growth hormone compound is a truncated version of hGH, i.e. one or more amino acid residues have been deleted from the N- and/or C-termini corresponding to SEQ No. 1 wherein the said compound retain desired biological properties of wild type hGH.

One embodiment of the present invention relates to a growth hormone compound comprising additional disulfide bonds in SEQ ID No. 1 or comprising one or more additional disulfide bond(s) compared to human growth hormone as defined in SEQ ID No. 1, wherein said compound has an in vitro activity which is comparable to the in vitro activity of hGH defined by SEQ ID NO 1. In vitro activity of growth hormone compounds is preferably measured in a BAF assay as described in Example 3 herein. In one embodiment a compound according to the invention may have an in vitro activity which is different from the in vitro acitivity of hGH. As described above a lower in vitro activity may be compensated by other in vivo functionalities. In an embodiment the in vitro activity may be such as at least 1%, such as at least 5%, for instance at least 10%, such as at least 25% of the activity of hGH. In a further embodiment the EC50 ratio for a compound relative to wild type hGH defined by SEQ ID No. 1 is not more that 10, not more than 8, not more than 6, not more than 4, not more than 2. In an embodiment the EC50 ratio for said compound compared to wild type hGH defined by SEQ ID No. 1 is from 5-0.01 or such as from 3-0.01 or such as is from 2-0.01. In an alternative the EC50 may according to the invention be measure by Surface Plasmon Resonance analysis (Biacore) as described in Example 4. In corresponding embodiments the in vitro activity determined by Biacore, may be such as at least 1%, such as at least 5%, for instance at least 10%, such as at least 25% of the activity of hGH. In further embodiments the EC50 ratio for a compound relative to wild type hGH defined by SEQ ID No. 1 determined by Biacore is not more that 10, not more than 8, not more than 6, not more than 4, not more than 2. In one embodiment the EC50 ratio for said compound compared to wild type hGH defined by SEQ ID No. 1 is from 5-0.01 or such as from 3-0.01 or such as 2-0.01.

Other examples of GH compounds into which additional disulphide bridges may be introduced include those disclosed in WO 92/09690 (Genentech), U.S. Pat. No. 6,004,931 (Genentech), U.S. Pat. No. 6,143,523 (Genentech), U.S. Pat. No. 6,136,536 (Genentech), U.S. Pat. No. 6,057,292 (Genentech), U.S. Pat. No. 5,849,535 (Genentech), WO 97/11178 (Genentech), WO 90/04788 (Genentech), WO 02/055532 (Maxygen APS and Maxygen Holdings), U.S. Pat. No. 5,951,972 (American Cynanamid Corporation), US 2003/0162949 (Bolder Biotechnologies, Inc.) which are incorporated herein by reference. Further included are natural variants of hGH, such as the 20 kDa described by Masuda, N et al, Biochim. Biophys. Acta 949 (1), 125-131 (1988).

In all embodiments described herein it is a further option that the growth hormone compound has a Gly residue in a position corresponding to position 120 of SEQ ID NO 1.

The present invention is also directed towards pharmaceutical compositions comprising growth hormone compounds as defined and described herein.

In one embodiment, pharmaceutical compositions of the present invention may be administered in several dosage forms, for example, as solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, tablets with co-formulation of absorption enhancing compounds, rinses, capsules, for example hard gelatine capsules and soft gelatine capsules, coated capsules, suppositories, drops, gels, sprays, powder, microparticles, nanoparticles, aerosols, inhalants, injection solution, in situ transforming solutions, for example in situ gelling, in situ setting, in situ precipitating, in situ crystallization, infusion solution, and implants.

In one embodiment of the present invention, the pharmaceutical compositions may be administered through oral, subcutaneous, intramuscular, nasal and i.v administration.

In one embodiment of the present invention, the oral pharmaceutical compositions may be administered through several routes of administration, for example, lingual, sublingual, buccal, in the mouth, rectal, in the stomach and intestine.

In one embodiment, pharmaceutical compositions of present invention are useful in the composition of solids, semisolids, powder and solutions for pulmonary administration of a peptide conjugate, such as e.g. a GH conjugate, using, for example a metered dose inhaler, dry powder inhaler and a nebulizer, all being devices well known to those skilled in the art.

In one embodiment, the pharmaceutical composition of the invention may further be compounded in, or attached to, for example through covalent, hydrophobic and electrostatic interactions, a drug carrier, drug delivery system and advanced drug delivery system in order to further enhance stability of the GH conjugate, increase bioavailability, increase solubility, decrease adverse effects, achieve chronotherapy well known to those skilled in the art, and increase patient compliance or any combination thereof. Examples of carriers, drug delivery systems and advanced drug delivery systems include, but are not limited to, polymers, for example cellulose and derivatives, polysaccharides, for example dextran and derivatives, starch and derivatives, chitosans and derivatives, poly(vinyl alcohol), acrylate and methacrylate polymers, polylactic and polyglycolic acid and block co-polymers thereof, polyethylene glycols, carrier proteins, for example albumin, gels, for example, thermo-gelling systems, for example block co-polymeric systems well known to those skilled in the art, micelles, liposomes, microspheres, nanoparticulates, liquid crystals and dispersions thereof, L2 phase and dispersions thereof, well known to those skilled in the art of phase behavior in lipid-water systems, polymeric micelles, multiple emulsions, self-emulsifying, self-microemulsifying, cyclodextrins and derivatives thereof, and dendrimers.

The various examples of delivery systems for oral formulation incorporated herein by reference include surfactants, which are known to increase the penetration of hydrophilic compounds. Examples of surfactants are; sodium caprate, tartaric acid, Brij56, Brij58, Brij35, Brij30, fatty acid sugars, sodium taurodeoxycholate, sodium dodecyl sulfate, p-t-octyl phenol poloxyethylene-9.5 (Triton X-100) as described by Takatsuka et al., Eur. J. Pharm. Biopharm. 62, 52-58 (2006). The oral delivery system may also include protease inhibitors and mucolytic substances. Examples of protease inhibitors are soybean trypsin inhibitor, aprotinin and chymostatin. Examples of mucolytic substances are dithiotreitol and N-acetyl cysteine. Enhancement of intestinal absorption of poorly absorbed hydrophilic compounds by simultaneous use of mycolytic agent and surfactant. Also the 5-CNAC and similar compounds developed by Emisphere (WO2008101240, WO200811283687, WO2008027854, WO2008014430, US20080095837).

The oral formulation delivery systems may also include tight junction modulators provide, which function as specific tight junction openers of epithelium cells. These tight junction modulators function both transient or non-transient and interfere with the protein complexes that hold the epithelium cells tightly together tight junctions (Kondoh et al., Mol Pharmacology 67, 749-756 (2005)). Other examples of the delivery system for oral formulation include mucoadhesive agents, for example thiol containing additives (co-formulation) or covalently attached sidechains can increase the adhesion to the mucos layer, chitosan and carbomer molecules, polyacrylates, PEG and its derivatives, (Palmberger et al., Eur. J. Pharm. Biopharm. 66, 405-412 (2007); Leitner, V. M et al., Eur. J. Pharm. Biopharm. 56, 207-214 (2003); H. L. Leuβen et al., Parm. Res. 13, 1668-1672 (1996); H. L. Leuβen et al., Int. J. Pharmaceuticals 141, 39-52 (1996); Takatsuka et al., Eur. J. Pharm. Biopharm. 62, 52-58 (2006). Additional examples of delivery systems for oral formulation include cavelolar/Lipid rafts, SMVT (sodium dependent multi vitamin transporter). Another examples of formulations for oral delivery includes receptor-mediated trancytosis such as IRF (intrinsic factor receptor) using Vitamin B12 (Cobalamin) as substrate, FcRn (neonatal Fc receptor) and Transferrin. (M. Gumbleton, Adv. Drug. Del. Rev. 49, 281-300 (2001); K. C. Partlow et al., Biomaterials 29, 3367-3375 (2008); (Lee et al., Biotechnol. Appl. Biochem. 46, 211-217 (2007); S. Y. Chae et al., Bioconjugate Chem. 19, 334-341 (2008); Russell-Jones G.: Chapter 17 in Membrane Transporters as Drug Targets (1999); Said and Mohammed Curr. Opin. Gastroent. 22, 140-146 (2006); Chalasani et al., J. Con. Release 117, 421-429 (2007); H. L1 & Z. M. Qian Med. Res. Rev. 22, 225-250 (2002); Liang & Yang Biochem. Biophys. Res. Comm. 225, 734-738 (2005).

In one embodiment the GH compounds of the present invention exert growth hormone activity and may be used for treating diseases or states which will benefit from an increase in the amount of circulating growth hormone. Such diseases or states include growth hormone deficiency (GHD); Turner Syndrome; Prader-Willi syndrome (PWS); Noonan syndrome; Down syndrome; chronic renal disease, juvenile rheumatoid arthritis; cystic fibrosis, HIV-infection in children receiving HAART treatment (HIV/HALS children); short children born short for gestational age (SGA); short stature in children born with very low birth weight (VLBW) but SGA; skeletal dysplasia; hypochondroplasia; achondroplasia; idiopathic short stature (ISS); GHD in adults; fractures in or of long bones, such as tibia, fibula, femur, humerus, radius, ulna, clavicula, matacarpea, matatarsea, and digit; fractures in or of spongious bones, such as the scull, base of hand, and base of food; patients after tendon or ligament surgery in e.g. hand, knee, or shoulder; patients having or going through distraction oteogenesis; patients after hip or discus replacement, meniscus repair, spinal fusions or prosthesis fixation, such as in the knee, hip, shoulder, elbow, wrist or jaw; patients into which osteosynthesis material, such as nails, screws and plates, have been fixed; patients with non-union or mal-union of fractures; patients after osteatomia, e.g. from tibia or 1st toe; patients after graft implantation; articular cartilage degeneration in knee caused by trauma or arthritis; osteoporosis in patients with Turner syndrome; osteoporosis in men; adult patients in chronic dialysis (APCD); malnutritional associated cardiovascular disease in APCD; reversal of cachexia in APCD; cancer in APCD; chronic abstractive pulmonal disease in APCD; HIV in APCD; elderly with APCD; chronic liver disease in APCD, fatigue syndrome in APCD; Chron's disease; impaired liver function; males with HIV infections; short bowel syndrome; central obesity; HIV-associated lipodystrophy syndrome (HALS); male infertility; patients after major elective surgery, alcohol/drug detoxification or neurological trauma; aging; frail elderly; osteo-arthritis; traumatically damaged cartilage; erectile dysfunction; fibromyalgia; memory disorders; depression; traumatic brain injury; subarachnoid haemorrhage; very low birth weight; metabolic syndrome; glucocorticoid myopathy; or short stature due to glucocorticoid treatment in children. Growth hormones have also been used for acceleration of the healing of muscle tissue, nervous tissue or wounds; the acceleration or improvement of blood flow to damaged tissue; or the decrease of infection rate in damaged tissue.

In one embodiment, the present invention relates to a method of treating diseases, wherein growth hormone compound activity maybe used for treating diseases or states which will benefit from an increase in the amount of circulating growth hormone compound said method comprising administering to a patient an effective amount of a pharmaceutical composition of growth hormone compound or its conjugate of SEQ ID No. 1.

In one embodiment, the present invention relates to a method comprising administration to a patient in need thereof an effective amount of a therapeutically effective amount of growth hormone compound according to the invention. The present invention thus provides a method for treating these diseases or states, the method comprising administering to a patient in need thereof a therapeutically effective amount of a growth hormone compound according to the present invention.

A “therapeutically effective amount” of a compound of the invention as used herein means an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease and its complications. An amount adequate to accomplish this is defined as “therapeutically effective amount”. Effective amounts for each purpose will depend on e.g. the severity of the disease or injury as well as the weight, sex, age and general state of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, which is all within the ordinary skills of a trained physician or veterinary.

In one embodiment, the invention provides the use of a growth hormone compound or its conjugate in the manufacture of a medicament used in the treatment of the above mentioned diseases or states.

The growth hormone compounds as defined and described herein in the present invention are intended to be used as a therapeutic protein.

The production of polypeptides is well known in the art. For example, polypeptides may be produced by classical peptide synthesis, e.g. solid phase peptide synthesis using tert-Boc or Fmoc chemistry or other well established techniques, see e.g. Greene and Wuts, “Protective Groups in Organic Synthesis”, John Wiley & Sons, 2006.

The polypeptides may also be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the polypeptide and capable of expressing the polypeptide in a suitable nutrient medium under conditions permitting the expression of the peptide. For polypeptides comprising non-natural amino acid residues, the recombinant cell should be modified such that the non-natural amino acids are incorporated into the polypeptide, for instance by use of tRNA mutants.

The polypeptides may also be produced using cell-free in vitro transcription/translation systems. A polypeptide containing novel unnatural amino acids may also be produced using frameshift or nonsense suppression systems e.g. as described in J. Am. Chem. Soc. 125, 11782-11783 (2003), Science 301, 964-967 (2003), Science 292, 498-500 (2001), Science 303, 371-373 (2004) and references herein.

The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The peptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration. For extra cellular products the proteinaceous components of the supernatant are isolated by filtration, column chromatography or precipitation, e.g. microfiltration, ultrafiltration, isoelectric precipitation, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, or the like, dependent on the type of polypeptide in question. For intracellular or periplasmic products the cells isolated from the culture medium are disintegrated or permeabilised and extracted to recover the product polypeptide or precursor thereof.

The DNA sequence encoding the polypeptide may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the peptide by hybridisation using specific DNA or RNA probes in accordance with standard techniques (see, for example, Sambrook, J, Fritsch, E F and Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequence encoding the polypeptide may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22, 1859-1869 (1981), or the method described by Matthes et al., EMBO Journal 3, 801-805 (1984). The DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or Saiki et al., Science 239, 487-491 (1988).

The DNA sequence encoding the peptide to be expressed may be inserted into any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

The vector may be an expression vector in which the DNA sequence encoding the polypeptide is operably linked to additional segments required for transcription of the DNA, such as a promoter. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA encoding the peptide to be expressed in a variety of host cells are well known in the art, cf. for instance Sambrook et al., supra.

The DNA sequence encoding the peptide to be expressed may also, if necessary, be operably connected to a suitable terminator, polyadenylation signals, transcriptional enhancer sequences, and translational enhancer sequences. The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.

The vector may also comprise a selectable marker, for instance a gene the product of which complements a defect in the host cell or one which confers resistance to a drug, for instance ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate. For large scale manufacture the selectable marker may for instance not be antibiotic resistance, e.g. antibiotic resistance genes in the vector may be excised when the vector is used for large scale manufacture. Methods for eliminating antibiotic resistance genes from vectors are known in the art, see e.g. U.S. Pat. No. 6,358,705 which is incorporated herein by reference.

To direct the peptide to be expressed into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro-sequence or presequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the peptide in the correct reading frame. Secretory signal sequences are commonly positioned 5′ to the DNA sequence encoding the peptide. The secretory signal sequence may be that normally associated with the peptide or may be from a gene encoding another secreted protein.

The procedures used to ligate the DNA sequences coding for the peptide to be expressed, the promoter and optionally the terminator and/or secretory signal sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., supra).

The host cell into which a DNA sequence or recombinant vector is introduced may be any cell which is capable of producing the present peptide and includes bacteria, yeast, fungi and higher eukaryotic cells. Examples of suitable host cells well known and used in the art are, without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines. The peptide to be expressed can also be produced by using in vitro transcription/translation systems commonly known in the art.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law).

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law

The invention as described herein is, without limitation hereto, further described by the following embodiments.

-   Embodiment 1. A growth hormone compound comprising one or more     additional disulfide bond(s) and at least one additional single     point mutation compared to human growth hormone as defined in SEQ ID     NO 1. -   Embodiment 2. A growth hormone compound according to embodiment 1,     wherein the growth hormone compound comprises an additional     disulfide bond connecting a loop segment and a helical segment or     within a loop segment or connecting loop segments or connecting     helical segments. -   Embodiment 3. A growth hormone compound according to embodiment 1 or     2, wherein the compound comprises at least one pair of mutations     corresponding to R16C/L117C, A17C/E174C, H21C/M170C, D26C/Y102C,     D26C/Y103C, N47C/T50C, Q49C/G161C, F54C/Y143C, F54C/S144C,     F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C,     I58C/S144C, P59C/Q137C, P61C/E66C, P61C/T67C, S71C/S132C,     L73C/S132C, L73C/F139C, R77C/I1380, R77C/F139C, L81C/Q141C,     L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C,     P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C,     L156C/F146C, L156C/T148C and/or V185C/S188C in SEQ ID NO 1. -   Embodiment 4. A growth hormone compound according to embodiment 3,     wherein the compound comprises at least one pair of mutations     corresponding to A170/E1740, H21C/M170C, D26C/V102C, D26C/Y103C,     F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C,     I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C,     L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C, F92C/T148C and/or     R94C/D107C in SEQ ID NO 1. -   Embodiment 5. A growth hormone compound according to any of the     previous embodiments, wherein the growth hormone compound comprises     an additional disulfide bond wherein at least one of the cysteines     is present in L3 corresponding to AA 128-154 in SEQ ID NO 1 or such     as in a region corresponding to AA 135-148 in SEQ ID NO 1. -   Embodiment 6. A growth hormone compound according to embodiment 5,     wherein at least one of the cysteines of the additional disulfide     bond is present in L3 in a position corresponding to AA 141, AA142,     AA143, AA144, AA145 or AA146, preferably AA143 or AA144 in SEQ ID NO     1. -   Embodiment 7. A growth hormone compound according to embodiment 6,     wherein the compound comprises at least one pair of mutations     corresponding to F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C,     S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C,     S71C/S132C, L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C,     L81C/Q141C, L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C,     S85C/S144C, P89C/F146C, F92C/F146C and/or F92C/T148C in SEQ ID NO 1. -   Embodiment 8. A growth hormone compound according to embodiment 7,     wherein the compound comprises at least one pair of mutations     corresponding to F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C,     S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C,     S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C and/or     F92C/T148C in SEQ ID NO 1. -   Embodiment 9. A growth hormone compound according to any of the     previous claims, wherein the growth hormone compound comprises an     additional disulfide bond connecting L3 with L1. -   Embodiment 10. A growth hormone compound according to embodiment 9,     wherein the compound comprises an additional disulfide bond     connecting an amino acid residue corresponding to AA54, AA55, AA56,     AA57, AA58 or AA59 in L3 with an amino acid corresponding to AA143     or AA144 in L1 of SEQ ID NO 1. -   Embodiment 11. A growth hormone compound according to embodiment 10,     wherein the compound comprises at least one pair of mutations     corresponding to F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C,     S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C and/or     S71C/S132C in SEQ ID NO 1. -   Embodiment 12 . A growth hormone compound according to any of     embodiments 1-8, wherein the growth hormone compound comprises an     additional disulfide bond connecting L3 with a helical segment. -   Embodiment 13. A growth hormone compound according to embodiment 12,     wherein the growth hormone compound comprises an additional     disulfide bond connecting L3 with helix 2. -   Embodiment 14. A growth hormone compound according to embodiment 13,     wherein the compound comprises an additional disulfide bond     connecting an amino acid residue corresponding to AA84 or AA85 in H2     with an amino acid corresponding to AA143 or AA144 in L3 of SEQ ID     NO 1. -   Embodiment 15. A growth hormone compound according to embodiment 12,     wherein the compound comprises at least one pair of mutations     corresponding to L73C/S132C, L73C/F139C, R77C/11380, R77C/F139C,     L81C/Q141C, L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C,     S85C/S144C, P89C/F146C, F92C/F146C and F92C/T148C in SEQ ID No. 1. -   Embodiment 16. A growth hormone compound according to embodiment 15,     wherein the compound comprises at least one pair of mutations     corresponding to L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C     and/or F92C/T148C in SEQ ID NO 1. -   Embodiment 17. A growth hormone compound according to any of     embodiments 1-8, wherein the growth hormone compound comprises an     additional disulfide bond connecting L2 with helix 1. -   Embodiment 18. A growth hormone compound according to embodiment 17,     wherein the compound comprises at least one pair of mutations     corresponding to D26C/V102C or D26C/Y103C. -   Embodiment 19. A growth hormone compound according to any of the     preceding embodiments, wherein the polypeptide sequence is at least     80%, such as 90%, such as 95%, such as 96%, such as 97%, or such as     98% identical to hGH defined by SEQ ID NO 1. -   Embodiment 20. A growth hormone compound according to any of the     preceding embodiments, wherein at least one additional single point     mutation is at a protease site. -   Embodiment 21

A growth hormone compound according to any of the preceding embodiments, wherein at least one additional single point mutation is at a position(s) corresponding to position 1-55, 57, 58, 60-63, 67-87, 89-91, 93, 95-100, 101,102-128, 131-132, 135-139, 141, 142, 144, 148-182, 184, 185 and/or 187-191 of SEQ ID NO 1.

-   Embodiment 22. A growth hormone compound according to any of the     preceding embodiments, wherein at least one additional single point     mutation is present in L1 (corresponding to AA 36-71 of SEQ ID NO 1)     and/or L3 (corresponding to AA 128-154 of SEQ ID NO 1). -   Embodiment 23. A growth hormone compound according to Embodiment 22,     wherein at least one additional single point mutation is present in     L1. -   Embodiment 24. A growth hormone compound according to Embodiment 23,     wherein at least one additional single point mutation is present in     the middle region of L1 (corresponding to AA 40-65 of SEQ ID NO 1). -   Embodiment 25. A growth hormone compound according to Embodiment 24,     wherein at least one additional single point mutation is present in     a position corresponding to position 40, 41, 42, 55, 57 and/or 62 of     SEQ ID NO 1. -   Embodiment 26. A growth hormone compound according to Embodiment 22,     wherein at least one additional single point mutation is present in     L3. -   Embodiment 27. A growth hormone compound according to Embodiments     26, wherein at least one additional single point mutation is present     in the middle region of L3 (corresponding to AA 135-148 of SEQ ID NO     1). -   Embodiment 28. A growth hormone compound according to Embodiment 27,     wherein at least one additional single point mutation is in a     position corresponding to position 134, 136, 139, 142 and/or 144 of     SEQ ID NO 1. -   Embodiment 29. A growth hormone compound according to any of the     preceding embodiments, wherein the in vitro activity for said     compound is at least 5% if the activity of wild type hGH defined by     SEQ ID NO 1. -   Embodiment 30. A growth hormone compound according to any of the     previous embodiments, wherein the functional in vivo half-life of     the polypeptide is 2 times or more compared to human growth hormone. -   Embodiment 31. A growth hormone compound according to any of the     previous embodiments, wherein the functional in vivo half-life of     the polypeptide is between 2 and 10 times more compared to human     growth hormone. -   Embodiment 32. A growth hormone compound according to any of the     preceding embodiments, wherein the growth hormone compound is     stabilized towards degradation by protease(s), such as digestive     proteases, such as pepsin, trypsin, chymotrypsin, carboxypeptidase     and/or elastases. -   Embodiment 33. A growth hormone compound according to embodiment 32,     wherein the compound is stabilized towards degradation by     Chymotrypsin and/or Elastase. -   Embodiment 34. A growth hormone compound according to embodiment 33,     wherein the compound comprises at least one pair of mutations     corresponding to H21C/M170C, D26C/V102C, D26C/Y103C, F54C/Y143C,     F54C/S144C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C,     I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C,     S85C/Y143C and/or S85C/S144C in SEQ ID NO 1. -   Embodiment 36. A growth hormone compound according to embodiment 33,     wherein the compound comprises at least one pair of mutations     corresponding to D26C/V102C, D26C/Y103C, S57C/Y143C, I58C/S144C,     P59C/Q137C, S71C/S132C, Q84C/Y143C, S85C/Y143C, S85C/S144C,     F92C/T148C and/or R94C/D107C in SEQ ID NO 1. -   Embodiment 38. A growth hormone compound according to embodiment 33,     wherein the compound comprises at least one pair of mutations     corresponding to S57C/Y143C, Q84C/Y143C, S85C/Y143C and/or     S85C/S144C in SEQ ID NO 1. -   Embodiment 40. A growth hormone compound according to any of the     previous embodiments, wherein the one or more additional disulfide     bond(s) is/are obtained by amino acid substitution of at least two     amino acid compared SEQ ID NO 1. -   Embodiment 41. The growth hormone conjugate according to any of the     previous embodiments wherein the growth hormone compound comprises     exactly one additional disulfide bond compared to SEQ ID NO 1. -   Embodiment 42. The growth hormone conjugate according to any of the     previous embodiments wherein the growth hormone compound comprises     at least two additional cysteines compared to human growth hormone     as defined in SEQ ID NO 1. -   Embodiment 43. The growth hormone conjugate according to any of the     previous embodiments wherein the compound comprises exactly 3 amino     acid substitutions compared to SEQ ID NO 1. -   Embodiment 44. The growth hormone conjugate according to any of the     previous embodiments comprising exactly two additional cysteines and     exactly one additional single point mutation compared to human     growth hormone as defined in SEQ ID NO 1. -   Embodiment 45. The growth hormone conjugate according to any of the     previous embodiments wherein the growth hormone compound comprises     exactly two additional cysteines compared to human growth hormone as     defined in SE Q ID NO 1. -   Embodiment 46. The growth hormone conjugate according to any of the     embodiments 1-44, wherein the growth hormone compound comprises     exactly three additional cysteines compared to human growth hormone     as defined in SE Q ID NO 1. -   Embodiment 47. The growth hormone conjugate according to embodiment     46, wherein the growth hormone compound comprises one additional     disulphide bond and an additional single cys mutation compared to     human growth hormone as defined in SE Q ID NO 1. -   Embodiment 48. The growth hormone conjugate according to embodiment     47, wherein the additional single cys mutation is selected from any     one of a single cys mutation in the N-terminal, H1, L1, H2, L2 or H3     regions of GH. -   Embodiment 49. The growth hormone conjugate according to embodiment     47, wherein the additional single cys mutation is located in a     position corresponding to AA 93-106, such as AA 99-106 or AA 99-103     in hGH. -   Embodiment 50. The growth hormone conjugate according to embodiment     47, wherein the additional single cys mutation is selected from the     group of mutation corresponding to: T3C, P5C, S7C, D11C, H180, Q29C,     E30C, E33C, A34C, Y35C, E88C, Q910, S95C, A98C, N99C, S1000, L1010,     V1020, Y1030, D1070, S1080, D1120, Q1220 and G1260 in hGH (SEQ ID     NO:1). -   Embodiment 51. A growth hormone compound according to any of the     preceding embodiments, wherein the growth hormone compound is     chemically modified by PEGylation, by attaching polymers, such as,     but not limited to sugar moieties, fatty acids, lipophilic groups,     albumin binders, vitamins, bile acids, spacers to the side chains or     main chain of the peptide. -   Embodiment 52. A growth hormone compound according to embodiment 51,     wherein chemical modification of growth hormone compound is     transient in nature, i.e. they may readily be removed in vivo. -   Embodiment 53. A growth hormone compound according to any of     embodiments 51-52, wherein chemical modifications of amino acid     residues can take place at the N-terminal of the peptide, at the     C-terminal of the peptide and/or between the N- and C-terminal of     the peptide. -   Embodiment 54. A growth hormone compound according to any of     embodiments 51-53, wherein the chemical modification takes place at     amino acid residues Phe1, GIn40, GIn141 or Phe191. -   Embodiment 55. A growth hormone compound according to any of     embodiments 51-54, wherein the chemical modification is by PEG and     wherein PEG is between 500 Da and 50 kDa. -   Embodiment 56. A growth hormone compound according to any of     embodiments 1-55, wherein the growth hormone compound is chemically     modified in order to facilitate transport across the epithelia. -   Embodiment 57. A growth hormone compound according to any of     embodiments 1-56, wherein the growth hormone compound is chemically     modified in order to facilitate transport across the epithelia when     compared to wthGH. -   Embodiment 58. A growth hormone compound according to any of     embodiments 1-57, wherein the growth hormone compound is chemically     modified in order to obtain a prolonged functional in vivo half-life     when compared to wthGH. -   Embodiment 59. A growth hormone compound according to embodiment 58,     wherein the functional in vivo half-life of said growth hormone     compound is 2 times or more compared to hGH. -   Embodiment 60. A growth hormone compound according to embodiment 59,     wherein the functional in vivo half-life is between 2 and 10 times     compared to hGH. -   Embodiment 61. A growth hormone compound according to any     embodiments 51-60, wherein the chemical modification takes place at     amino acid residues not interfering with binding of the growth     hormone compound to the hGHR. -   Embodiment 62. A growth hormone compound according to any of     embodiments 55-61, wherein the growth hormone compound is stabilized     towards proteolytic degradation by protease(s), such as as digestive     proteases, such as pepsin, trypsin, chymotrypsin, carboxypeptidase     and/or elastases. -   Embodiment 63. A method for preparing a growth hormone compound with     increased stability towards proteolytic degradation, which method     comprises a step of     -   a. introducing one or more additional disulfide bond(s) and at         least one additional single point mutation, compared to human         growth hormone (hGH) as defined in SEQ ID No. 1, in said growth         hormone compound. -   Embodiment 64. A method for preparing a growth hormone compound with     increased stability towards proteolytic degradation according to     embodiments 63 which method comprises a step of     -   a. introducing one or more additional disulfide bond(s) by         substituting one or more amino acid residue(s) with one or more         cysteine(s) compared to human growth hormone (hGH) and     -   b. introducing at least one additional single point mutation(s)         by substituting one or more amino acid residue(s) compared to         human growth hormone (hGH). -   Embodiment 65. A method for preparing a growth hormone compound with     increased stability towards proteolytic degradation according to     embodiment 64, which comprises steps of     -   a. introducing one or more additional disulfide bonds in hGH by         adding one or more cysteine residue(s) compared to human growth         hormone (hGH) and     -   b. introducing at least one additional single point mutation(s)         by substituting one or more amino acid residue(s) compared to         human growth hormone (hGH). -   Embodiment 66. A pharmaceutical composition comprising growth     hormone compound according to any of embodiments 1 to 62 and a     pharmaceutically acceptable carrier/s. -   Embodiment 67. A pharmaceutical composition according to embodiment     66, wherein said composition can be administered through lingual,     sublingual, buccal, in the mouth, oral, in the stomach and     intestine, nasal, pulmonary, epidermal, dermal, transdermal, and     parenteral to patients. -   Embodiment 68. A pharmaceutical composition according to embodiment     66 or 67, wherein said composition is orally administered     composition. -   Embodiment 69. A method of preparing a pharmaceutical composition     wherein said composition comprising of growth hormone compound     according to any of embodiments 1 to 62 and a pharmaceutically     acceptable carrier/s. -   Embodiment 70. A method of treating diseases or states where a     patient benefits from an increase in the amount of circulating     growth hormone compound said method comprising administering to     patient an effective amount of a growth hormone compound according     to any of embodiments 1 to 62 or a pharmaceutical composition     according to any of embodiments 66-68. -   Embodiment 71. A method of treating diseases according to     embodiments 69 or 70, wherein disease is selected from growth     hormone deficiency (GHD); Turner Syndrome; Prader-Willi syndrome     (PWS); Noonan syndrome; Down syndrome; chronic renal disease,     juvenile rheumatoid arthritis; cystic fibrosis, HIV-infection in     children receiving HAART treatment (HIV/HALS children); short     children born short for gestational age (SGA); short stature in     children born with very low birth weight (VLBW) but SGA; skeletal     dysplasia; hypochondroplasia; achondroplasia; idiopathic short     stature (ISS); GHD in adults; fractures in or of long bones, such as     tibia, fibula, femur, humerus, radius, ulna, clavicula, matacarpea,     matatarsea, and digit; fractures in or of spongious bones, such as     the scull, base of hand, and base of food; patients after tendon or     ligament surgery in e.g. hand, knee, or shoulder; patients having or     going through distraction oteogenesis; patients after hip or discus     replacement, meniscus repair, spinal fusions or prosthesis fixation,     such as in the knee, hip, shoulder, elbow, wrist or jaw; patients     into which osteosynthesis material, such as nails, screws and     plates, have been fixed; patients with non-union or mal-union of     fractures; patients after osteatomia, e.g. from tibia or 1st toe;     patients after graft implantation; articular cartilage degeneration     in knee caused by trauma or arthritis; osteoporosis in patients with     Turner syndrome; osteoporosis in men; adult patients in chronic     dialysis (APCD); malnutritional associated cardiovascular disease in     APCD; reversal of cachexia in APCD; cancer in APCD; chronic     abstractive pulmonal disease in APCD; HIV in APCD; elderly with     APCD; chronic liver disease in APCD, fatigue syndrome in APCD;     Chron's disease; impaired liver function; males with HIV infections;     short bowel syndrome; central obesity; HIV-associated lipodystrophy     syndrome (HALS); male infertility; patients after major elective     surgery, alcohol/drug detoxification or neurological trauma; aging;     frail elderly; osteo-arthritis; traumatically damaged cartilage;     erectile dysfunction; fibromyalgia; memory disorders; depression;     traumatic brain injury; subarachnoid haemorrhage; very low birth     weight; metabolic syndrome; glucocorticoid myopathy; or short     stature due to glucocorticoid treatment in children. -   Embodiment 72. A growth hormone according to any of embodiments 1 to     62 for use as an medicament. -   Embodiment 74. A growth hormone according to any of embodiments 1 to     62 for use as an medicament for treatment of a disease is selected     from: growth hormone deficiency (GHD); Turner Syndrome; Prader-Willi     syndrome (PWS); Noonan syndrome; Down syndrome; chronic renal     disease, juvenile rheumatoid arthritis; cystic fibrosis,     HIV-infection in children receiving HAART treatment (HIV/HALS     children); short children born short for gestational age (SGA);     short stature in children born with very low birth weight (VLBW) but     SGA; skeletal dysplasia; hypochondroplasia; achondroplasia;     idiopathic short stature (ISS); GHD in adults; fractures in or of     long bones, such as tibia, fibula, femur, humerus, radius, ulna,     clavicula, matacarpea, matatarsea, and digit; fractures in or of     spongious bones, such as the scull, base of hand, and base of food;     patients after tendon or ligament surgery in e.g. hand, knee, or     shoulder; patients having or going through distraction oteogenesis;     patients after hip or discus replacement, meniscus repair, spinal     fusions or prosthesis fixation, such as in the knee, hip, shoulder,     elbow, wrist or jaw; patients into which osteosynthesis material,     such as nails, screws and plates, have been fixed; patients with     non-union or mal-union of fractures; patients after osteatomia, e.g.     from tibia or 1st toe; patients after graft implantation; articular     cartilage degeneration in knee caused by trauma or arthritis;     osteoporosis in patients with Turner syndrome; osteoporosis in men;     adult patients in chronic dialysis (APCD); malnutritional associated     cardiovascular disease in APCD; reversal of cachexia in APCD; cancer     in APCD; chronic abstractive pulmonal disease in APCD; HIV in APCD;     elderly with APCD; chronic liver disease in APCD, fatigue syndrome     in APCD; Chron's disease; impaired liver function; males with HIV     infections; short bowel syndrome; central obesity; HIV-associated     lipodystrophy syndrome (HALS); male infertility; patients after     major elective surgery, alcohol/drug detoxification or neurological     trauma; aging; frail elderly; osteo-arthritis; traumatically damaged     cartilage; erectile dysfunction; fibromyalgia; memory disorders;     depression; traumatic brain injury; subarachnoid haemorrhage; very     low birth weight; metabolic syndrome; glucocorticoid myopathy; and     short stature due to glucocorticoid treatment in children. -   Embodiment 73. Use of a growth hormone according to any of     embodiments 1 to 62 as an medicament. -   Embodiment 74. Use of growth hormone compound according to any of     embodiments 1 to 62 in treatment of disease. -   Embodiment 75. Use according to embodiment 73 or embodiment 74,     wherein the disease is selected from growth hormone deficiency     (GHD); Turner Syndrome; Prader-Willi syndrome (PWS); Noonan     syndrome; Down syndrome; chronic renal disease, juvenile rheumatoid     arthritis; cystic fibrosis, HIV-infection in children receiving     HAART treatment (HIV/HALS children); short children born short for     gestational age (SGA); short stature in children born with very low     birth weight (VLBW) but SGA; skeletal dysplasia; hypochondroplasia;     achondroplasia; idiopathic short stature (ISS); GHD in adults;     fractures in or of long bones, such as tibia, fibula, femur,     humerus, radius, ulna, clavicula, matacarpea, matatarsea, and digit;     fractures in or of spongious bones, such as the scull, base of hand,     and base of food; patients after tendon or ligament surgery in e.g.     hand, knee, or shoulder; patients having or going through     distraction oteogenesis; patients after hip or discus replacement,     meniscus repair, spinal fusions or prosthesis fixation, such as in     the knee, hip, shoulder, elbow, wrist or jaw; patients into which     osteosynthesis material, such as nails, screws and plates, have been     fixed; patients with non-union or mal-union of fractures; patients     after osteatomia, e.g. from tibia or 1st toe; patients after graft     implantation; articular cartilage degeneration in knee caused by     trauma or arthritis; osteoporosis in patients with Turner syndrome;     osteoporosis in men; adult patients in chronic dialysis (APCD);     malnutritional associated cardiovascular disease in APCD; reversal     of cachexia in APCD; cancer in APCD; chronic abstractive pulmonal     disease in APCD; HIV in APCD; elderly with APCD; chronic liver     disease in APCD, fatigue syndrome in APCD; Chron's disease; impaired     liver function; males with HIV infections; short bowel syndrome;     central obesity; HIV-associated lipodystrophy syndrome (HALS); male     infertility; patients after major elective surgery, alcohol/drug     detoxification or neurological trauma; aging; frail elderly;     osteo-arthritis; traumatically damaged cartilage; erectile     dysfunction; fibromyalgia; memory disorders; depression; traumatic     brain injury; subarachnoid haemorrhage; very low birth weight;     metabolic syndrome; glucocorticoid myopathy; or short stature due to     glucocorticoid treatment in children. -   Embodiment 76. Use of growth hormone compound according to any of     embodiments 1 to 62 in manufacture of a medicament to be used in     treatment of growth hormone deficiency (GHD); Turner Syndrome;     Prader-Willi syndrome (PWS); Noonan syndrome; Down syndrome; chronic     renal disease, juvenile rheumatoid arthritis; cystic fibrosis,     HIV-infection in children receiving HAART treatment (HIV/HALS     children); short children born short for gestational age (SGA);     short stature in children born with very low birth weight (VLBW) but     SGA; skeletal dysplasia; hypochondroplasia; achondroplasia;     idiopathic short stature (ISS); GHD in adults; fractures in or of     long bones, such as tibia, fibula, femur, humerus, radius, ulna,     clavicula, matacarpea, matatarsea, and digit; fractures in or of     spongious bones, such as the scull, base of hand, and base of food;     patients after tendon or ligament surgery in e.g. hand, knee, or     shoulder; patients having or going through distraction oteogenesis;     patients after hip or discus replacement, meniscus repair, spinal     fusions or prosthesis fixation, such as in the knee, hip, shoulder,     elbow, wrist or jaw; patients into which osteosynthesis material,     such as nails, screws and plates, have been fixed; patients with     non-union or mal-union of fractures; patients after osteatomia, e.g.     from tibia or 1st toe; patients after graft implantation; articular     cartilage degeneration in knee caused by trauma or arthritis;     osteoporosis in patients with Turner syndrome; osteoporosis in men;     adult patients in chronic dialysis (APCD); malnutritional associated     cardiovascular disease in APCD; reversal of cachexia in APCD; cancer     in APCD; chronic abstractive pulmonal disease in APCD; HIV in APCD;     elderly with APCD; chronic liver disease in APCD, fatigue syndrome     in APCD; Chron's disease; impaired liver function; males with HIV     infections; short bowel syndrome; central obesity; HIV-associated     lipodystrophy syndrome (HALS); male infertility; patients after     major elective surgery, alcohol/drug detoxification or neurological     trauma; aging; frail elderly; osteo-arthritis; traumatically damaged     cartilage; erectile dysfunction; fibromyalgia; memory disorders;     depression; traumatic brain injury; subarachnoid haemorrhage; very     low birth weight; metabolic syndrome; glucocorticoid myopathy; or     short stature due to glucocorticoid treatment in children.

EXAMPLES

The invention will be further defined by reference to the following examples, which describe the preparation and characterization of the various compounds described herein and methods for assaying their biological activity. It will be apparent to those skilled in the art that many modifications, both to the materials and methods may be practiced without departing from the scope of the invention.

Example 1 General Method for Preparing a gGH Compounds

The gene coding for the growth hormone compound is inserted recombinantly into a plasmid vector. Mutations are introduced by using QuikChange site-directed mutagenesis kit (Stratagene). A suitable E. coli strain is subsequently transformed using the plasmid vector. Protein is expressed as soluble protein with an N-terminal Histidine rich peptide tag suitable for immobilised metal affinity chromatography purification.

Cell stock are prepared in 50% glycerol and stored at −80° C. Glycerol stock strain is inoculated into LBA plates and subsequently incubated at 37° C. overnight. The content of each plate is washed with LB medium and diluted into 500 ml LB+AMP medium for expression. The cultures are incubated at 37° C. with shaking at 220 rpm until OD₆₀₀ 0.6 is reached. Succeeding induction is performed using 0.2 mM IPTG at 30° C. for 6 hours, giving a final OD₆₀₀ of 2.0. Cells are finally harvested by centrifugation.

Cells are subsequently suspended in 20 mM Tris-HCl, pH 8.5 and disrupted using a cell disrupter at 30 kPSI. The supernatant is collected by centrifugation and subsequently subjected to chromatographic purification.

The purification is performed using immobilised metal affinity chromatography as capturing step, followed by removal of the peptide tag using di-amino-peptidase from Unizyme. Final purification is achieved by ion-exchange chromatography. The purification could also be achieved by using but not limited to ion-exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, size exclusion chromatography and membrane based separation techniques known to a person skilled in the art.

PEG groups may be attached to the N-terminal by reacting the hGH compound with 2 equivalents of e.g. Peg-5000-aldehyde (RAPP Polymere, 12 5000-6). Reaction maybe initiated by addition of NaCNBH₃ in 0.5 ml MeCN in 10 steps and the reaction mixture left for 20 h. PEG groups may be attached to Q40 by first reacting the hGH compound with 1,3-diamino-2-propanol (Fluka 33262) utilising microbiel transglutaminase as catalyst (see below).

Purification of Cys mutated GH protein may require modification to the standard procedure. The method is exemplified using MEAE-hGH[Q84C, Y143C, L101C]. Alternative mutants may be isolated and purified in a similar way possible with minor modification to the isolation and purification scheme based on common general knowledge in the art.

Cells are harvested by centrifugation (6056×G for 30 min) and may be stored at −80° C. Homogenization: Cell pellets (2580 g) were dissolved in ca. 20 kg buffer (20 mM Tris, 0.1% Tween-20, 5 mM EDTA, pH=8.5) and pH adjusted to 8.5 followed by homogenization with two pressures drops 1000 and 200 bar. The cell homogenate (˜20 kg) was diluted 1:1 in 8M urea, resulting in a 4M urea concentration. To this solution was added cystamine 2HCl (18 g) (0.9 g/L) and the resulting mixture incubated over night at 5° C. with gentle stirring. Filtration: The homogenate was filtered by dead end filtration 1.0 and 0.5 μm resulting in approx. 42 kg permeate. Purification of MEAE-hGH[Q84C, Y143C, L101C] consists of three chromatographic steps and an enzyme digestion to remove the N-terminal MEAE-sequence. MEAE-hGH[Q84C, Y143C, L101C] was captured using Q Sepharose XL (GE Healthcare) and eluted by linear gradient elution in a Tris-NaCl buffer, pH 8.5. To the collected pool was added 1.0 M NaCl before loading onto Phenyl Sepharose 6FF (high sub) (GE Healthcare). Step elution in WFI was used to elute the protein and lower the conductivity for enzyme digestion.

The MEAE-sequence was removed using the enzyme DAPI (dipeptidylaminopeptidase 1). DAPI cleaves two amino acids at a time and stops after AE as the second amino acid in hGH is a proline, which is a natural stop sign for DAPI. Digestion was carried out at 40° C. at a protein concentration of 2 mg/mL, pH 4.3. The reaction was followed by LC-MS and stopped after approx. 60 min when digestion was complete. After cooling to 5° C., 39% v/v cold ethanol (99%) was added to iso-precipitate the protein and pH was adjusted to 4.9. The reaction mixture was stored at 5° C. for at least 2 hrs. to precipitate the protein.

Precipitated hGH[Q84C, Y143C, L101C] was re-dissolved in a 7 M ureatriethanolamine buffer, pH 7.5, and purified on Source 30Q (GE Healthcare) to separate target protein from minor amounts of un-cleaved material and other impurities. Final product is eluted by linear gradient elution in a triethanolamine buffer, pH 7.5. Product purity is >97% by RP-HPLC analysis (UV 214 nm).

Coupling of Transaminated and Oxidised hGH Compound (I) with a mPEG-group.

The following solutions were prepared:

-   Buffer A: Triethanolamine (119 mg, 0.8 mmol) was dissolved in water     (40 mL) and pH adjusted to 8.5. -   Buffer B: 20 mM Triethanolamine; 0.2 M NaCl.

(A) Transamination of hGH (III) with 1,3-diamino-2-propanol

hGH (8.64 g) was dissolved in Buffer A (500 mL) with stirring. To this solution was added slowly a mixture of 1,3-diamino-2-propanol (DAP) (8.1 g, Fluke 33262) in Buffer A (50 mL). pH of the resulting mixture was adjusted to 8.5 by addition of aq. HCl. mTGase (2.8 mL, 1.3 mg/mL)) was added while mixing. The final mixture was stirred overnight at RT. The reaction mixture was diluted with buffer A (1.2 L) and the product was purified by ion exchange chromatography. 100 mL/min-200 mL/frac. Step Buffer B 40%—gradient 40-100% Buffer B over 15 CV=225 min.

(B) Oxidation of Transaminated hGH

-   Buffer A: Triethanolamine (119 mg, 0.8 mmol) was dissolved in water     (40 mL) and pH adjusted to 8.5. -   Buffer B: 3-methylthio-1-propanol (725 mg, 7.1 mmol) was dissolved     in Buffer A (10 mL). -   Buffer C: HEPES (5.96 g) was dissolved in water (1.0 L) and pH     adjusted to 7.0 -   Periodate: NalO₄ (48.1 mg, 0.225 mmol) was dissolved in water (1.0     mL).

To a solution of DAP reacted hGH (10 mg, 0.5 μmol) was added Buffer B (0.2 mL) followed by the periodate solution (0.03 mL). After 20 min's of cold incubation the mixture was dialyzed 4 times with buffer C. The residue was concentrated to 1 mL.

(C) Reductive Amination of Oxidised hGH with PEG-Reagent.

The final solution from (B) (1 mL, 0.45 μmol) was mixed with a PEG-amine solution (2 mL, 0.3 μmol) in 25 mM HEPES buffer pH 7.0 and the resulting mixture was slowly rotated at room temperature for 1 hr. After 1 hr NaCNBH₃ (100 μL of a solution of NaCNBH₃ (20 mg) in water (0.5 mL)) was added portion wise (10×). The mixture was kept at room temperature in the dark for 18-24 hours. The mixture was purified on a MonoQ, buffer changed and concentrated. mPEG-amine reagents are commercially available.

Example 2 Protein Chemical Characterization of Purified Growth Hormone Compounds

The intact purified protein was analysed using MALDI-MS. The observed mass corresponded to the theoretical mass deduced from the amino acid sequence.

The expected linkage of the three disulfide bonds in each compound was demonstrated by peptide mapping using trypsin and AspN digestion followed by MALDI-MS analysis of the digest before and after reduction of the disulfide bonds with DTT.

Example 3 Analysis of the Biological Activity of the Purified Growth Hormone Compounds

The biological activity of hGH compounds was measured in a cell based receptor potency proliferation assay, namely a BAF assay. The method is general for the hGH compounds.

The BAF-3 cells (a murine pro-B lymphoid cell line derived from the bone marrow) is IL-3 dependent for growth and survival. IL-3 activates JAK-2 and STAT which are the same mediators GH is activating upon stimulation.

The BAF-3 cells may be transfected with a plasmid containing the hGH receptor. Clones able to proliferate upon stimulation with hGH may be turned into hGH-dependent cell lines hereafter referred to as BAF3-GHR. The cell lines respond to GH with a dose-related growth pattern and can therefore be used to evaluate the effect of different hGH compounds in a proliferation assay.

The BAF-3 GHR cells are grown in starvation medium (culture medium without GH) for 24 hours at 37° C., 5% CO₂. The cells are centrifuged, the medium is removed and the cells are resuspended in starvation medium to 2.22×10⁵ cells/ml. Portions of 90 μl of the cell supernatant are seeded into microtiter plates (96 well NUNC-clone). Different concentrations of growth hormone compound are added to the cells, and the plates are incubated for 72 hours at 37° C., 5% CO₂.

AlamarBlue is a redox indicator, AlamarBlue® (BioSource cat no Dal 1025) which is reduced by reactions innate to cellular metabolism and, therefore, provides an indirect measure of viable cell number. The AlamarBlue® is diluted 6 times (5 μl AlamarBlue®+25 μl stavation medium) and 30 μl of the diluted AlamarBlue® is added to each well. The cells are then incubated for another 4 hours. Finally the metabolic activity of the cells is measure in a fluorescence plate reader using an excitation filter of 544 nM and an emission filter of 590 nM.

The result for a given compound is expressed as the ratio between EC₅₀ of said compound and the EC₅₀ of wthGH run in parallel. Further results are given in table 6 below.

TABLE 2 EC50 values for hGH compounds relative to EC50 value for hGH Cys Compound Average Dev A17C-E174C 0.6 0.1 H21C-M170C 0.4 0.3 R94C-D107C 0.8 0.4 Q84C-Y143C 0.3 0.1 S71C-S132C 0.24 0.02 All hGH compounds tested were equipotent with or more potent than wthGH.

TABLE 2A EC50 values for hGH compounds with a PEG group relative to EC50 value for hGH. EC50 BAF RATIO COMPOUND (hGH compound/hGH wt) HGH 1.0 HGH (Q84C, Y143C) Q40-PEG5000 0.75 HGH (Q84C, Y143C) N-TERM PEG750 0.475 HGH (Q84C, Y143C) N-TERM PEG5000 1.1475

Example 3A In Vivo Dose-response Study in Hypophysectomised Sprague Dawley Rats

The in vivo dose-response relationship may be studied in hypophysectomised male Sprague Dawley rats. The hypophysectomised rat is a well known and recognised animal model of growth hormone deficiency, where no production of growth hormone occurs after the surgical removal of the pituitary gland. This also leads to low circulating levels of insulinlike growth factor-1 (IGF-1) another important clinical feature of growth hormone deficiency in humans.

The following describes the assay performed using hGH and one hGH variant. Alternative variants and compounds may be tested using similar procedure.

Hypophysectomy was performed on 4 week old male rats weighing 90-100 g. The animals entered the study 3-4 weeks after the surgery weighing 100-110 g. Animals with a body weight gain of more than 10% during the 3-4 weeks after surgery were not allowed to enter the study.

Seventy hypophysectomised Sprague Dawley rats were randomly allocated to seven dosing groups with ten animals in each group. One group received vehicle only and served as an untreated control group. Three groups received test compound (hGH Q84C, Y143C) 33, 3.3 and 0.33 nmol respectively and three groups received hGH as a comparator 50, 5.0 and 0.5 nmol respectively. Both compounds and vehicle were administered as a single subcutaneous dose in the neck. The body weight was measured daily between 8-10 am for one week.

Both hGH Q84C, Y143C and hGH induced a dose-dependent increase in body weight when body weight on Day 0 was compared to that of Day 7.

A sigmoidal dose-response equation was fitted to the experimental data (increase in body weight between Day 0-7) by non-linear regression analysis in order to calculate parameter estimates of E_(max), E₀ and ED₅₀. The equation was a sigmoidal dose-response built-in equation in GraphPad Prism version 4.00 for Windows (GraphPad Software Inc., San Diego, USA). Data including parameter estimates and 95% confidence intervals are presented in Table 3.

No difference in parameter estimates of E₀ and E_(max) was observed for hGH Q84C, Y143C and hGH. However ED₅₀ was significantly lower for hGH Q84C, Y143C compared to hGH indicating an increased in vivo potency of hGH Q84C, Y143C.

TABLE 3 The response as increase in body weight on Day 7 compared to Day 0 was fitted to a sigmoidal dose-response equation in order to estimate E_(max), E₀ and ED₅₀. hGH Q84C, Y143C hGH wt E₀ (g) 0.0  0.2  (−1.9-1.8)  (−1.7-2.0)  ED₅₀ (nmol) 0.29 0.70 (0.20-0.41) (0.50-0.99) E_(max) (g) 26.3  27.5  (24.8-27.9) (25.9-29.1) Mean (95% confidence interval)

Example 4 Receptor Interaction Studies by Surface Plasmon Resonance Analysis

Receptor interaction of hGH compounds was analyzed using surface plasmon resonance analysis. The method is general for the hGH compounds and is exemplified by the Q84C/Y143C hGH compound.

The interaction of hGH and analogues with hGH binding protein (hGHBP) was studied by surface plasmon resonance using either a Proteon instrument (BioRad) or a T100 Biacore (GE Health Care, Sweden). Anti-hGH mAb (Fitzgerald Industries International, USA, #10G05B) was covalently immobilized onto a carboxylated matrix by amine coupling according to manufacturers instruction at a level of typically 5000-9000 RU. wthGH or analogues were captured at 9-25 μg/ml in running buffer (10 mM HEPES, 0.15 M NaCl, 30 mM EDTA, 0.05% Surfactant P20, pH 7.4), which typically resulted in 100-400 RU captured ligand. hGHBP at a concentration of 0-800 nM was subsequently injected over the surface at 30-100 ml/min. A surface with immobilized anti-hGH mAb but without captured hGH was used as reference.

Kinetic data was analyzed with appropiate software provided by the instrument manufactuar with the 1:1 Langmuir binding model.

Analysis showed (Table 4) that hGH Q84C, Y143C had similar or slightly higher affinity towards growth hormone binding protein than wthGH and that hGH Q84C, Y143C was equipotent with wild type hGH within the experimental error. Additional analysis of further hGH variants is included in Table 7.

TABLE 4 Ec50 ± std ka (1/Ms) kd (1/s) KD (nM) dev. (nM) hGH wt 1.9 × 10⁵ 6.1 × 10⁻⁴ 3.3 0.7 ± 0.3 hGH Q84C, Y143C 2.0 × 10⁵ 4.8 × 10⁻⁴ 2.5 0.3 ± 0.1

Example 5 Assay for Measuring Rate of Protease Degradation of Wild Type hGH and GH Compounds

The compound of interest is digested by a relevant protease (Trypsin, Chymotrypsin, Pepsin, Elastase, Factor Vila, Factor Xa, Proteinase K, Carboxy peptidase, DPPIV, Neutral Endopeptidase, Granzyme B, Proline-endopeptidase, Staphylococcal peptidase I, Thermolysin, Thrombin, Arg-C proteinase, Asp-N endopeptidase, Caspase 1-10, Clostripain, Enterokinase, Glutamyl endopeptidase, Granzyme B, LysC, LysN, Proline-endopeptidase and Staphylococcal peptidase I or tissue extracts) in an appropriate buffer (e.g. PBS or ammonium bicarbonate) at 37° C. for up till 24 hours. Proteolytic degradation is assessed by a HPLC assay.

General Method

Proteolytic Digestion:

100 μL of test compound solution at 1 mg/ml in ammonium bicarbonate buffer is degraded by enzyme for up till 24 hours at 37° C. Sub-samples are taken to various time points and the proteolytic reaction is stopped by acidifying the sample by 10 times dilution into 1% TFA. These diluted samples are analysed by reversed phase HPLC to estimate the degree of proteolytic digestion.

HPLC Method:

10 μL of the above solution is injected on a reversed phase Vydac C4 2×150 mm column eluted with a linear gradient from 0.1% TFA in water to 100% acetonitrile containing 0.1% TFA over a period of 30 min at a flow rate of 0.2 ml/min. Detection of peaks is performed at 214 nm UV absorption. % intact compound at time point t=T is calculated from the peak area at time point t=T (A_(T)) and the peak area at t=0 (A₀) as (A_(T)/A₀)×100%. The results provided in table 6 here below were obtained after 4 hours (T=4 in above equation).

% intact compound is plotted against time using GraphPad Prims software ver. 5.01. T½ is calculated as one phase decay also by GraphPad Prism software.

Enzymes used in the example are elastase (135 ng, Sigma from porcine pancrease) and chymotrypsin (13 ng, Roche sequencing grade). Buffer is 50 mM ammonium bicarbonate pH 8.5.

TABLE 5 T½ (hours) for degradation of wild type hGH and hGH variants by chymotrypsin and elastase. chymotrypsin elastase Compound T½ T½ hGH wt 3.6 1.6 hGH A17C, E174C 7.5 hGH H21C, M170C 22 hGH R94C, D107C 2.5 hGH Q84, Y143C N/A 6.2

Example 6 Analysis of Selected Compounds by BAF Assay and Proteolytic Digestion as Described in Example 3 and 5

TABLE 6 Analysis of growth hormone compounds comprising additional disulfide bonds. Chymotrypsin Elastase Domains EC50 BAF stability stability linked by Ratio (hGH Intact Intact additional compound/ compound compound disulfide Compound hGH) (%) (%) bond HGH 1.0 42 25 HGH (A17C 0.6 45 10 H1-H4 E174C) HGH (H21C 0.5 72 10 H1-H4 M170C) HGH (D26C, 0.5 55 65 H1-L2 V102C) HGH (D26C, 0.5 55 45 H1-L2 Y103C) HGH (F54C, 0.6 55 20 L1-L3 Y143C) HGH (F54C, 0.5 60 20 L1-L3 S144C) HGH (F54C, 0.6 40 25 L1-L3 F146C) HGH (S55C, 0.5 90 25 L1-L3 Y143C) HGH (S57C, 0.3 75 50 L1-L3 Y143C) HGH (I58C 0.7 70 25 L1-L3 Q141C) HGH (I58C, 0.6 55 20 L1-L3 Y143C) HGH (I58C, 1.2 65 30 L1-L3 S144C) HGH (P59C 0.7 72 35 L1-L3 Q137C) HGH (S71C 0.2 90 45 L1-L3 S132C) HGH (L81C, 0.7 85 15 H2-L3 Y143C) HGH (Q84C 0.5 100 80 H2-L3 Y143C) HGH (S85C 0.5 80 70 H2-L3 Y143C) HGH (S85C, 0.7 81 60 H2-L3 S144C) HGH (F92C, 0.6 40 55 H2-L3 T148C) HGH (R94C 0.8 38 70 H2-H3 D107C)

Example 7 Analysis of Selected Compounds by BAF Assay, SPR-analysis and Proteolytic Digestion as Described in Example 3, 4 and 5

TABLE 7 Analysis of growth hormone compounds comprising an additional disulfide bonds in combination with an additional single point mutation. Chymotrypsin Elastase EC50 BAF stability stability Ratio (hGH Intact Intact SPR Kd compound/ compound compound Compound (nM) hGH) (%) (%) HGH 3.3 1.00 40 20 HGH (Q84C, 2.5 0.66 90 70 Y143C) HGH (S62N) 1.0 1.83 50 70 HGH (S62Q) 2.0 1.02 50 70 HGH (Q84C, 4.5 1   85 45 Y143C, S55Q) HGH (Q84C, 5.0 1.69 95 20 Y143C, S55E) HGH (Q84C, 1.16 90 75 Y143C, S55N) HGH (Q84C, 1.07 90 85 Y143C, S55T) HGH (Q84C, 2.0 1.18 90 65 Y143C, S57Q) HGH (Q84C, 2.0 0.84 84 75 Y143C, S57E) HGH (Q84C, 0.82 85 85 Y143C, S57N) HGH (Q84C, 1.99 95 60 Y143C, S57H) HGH (Q84C, 2.0 0.29 80 50 Y143C, S62T) HGH (Q84C, 2.5 0.80 90 90 Y143C, S62N) HGH (Q84C, 4.0 ND 90 70 Y143C, S62C) HGH (Q84C, 2.0 0.85 90 90 Y143C, S62Q) HGH (Q84C, 2.0 ND 80 75 Y143C, S62H) HGH (Q84C, 3.0 ND 85 80 Y143C, S62E) HGH (Q84C,  3.00 0.99 100 75 Y143C, L101C) HGH (Q84C, 2.0/2.5 2.19 90 50 Y143C, R134V) HGH (Q84C, 2.0/2.5 0.94 80 50 Y143C, G136V) HGH (Q84C, 2.5 0.55 80 65 Y143C, T142N) HGH (Q84C, 4.0 0.60 85 45 Y143C, S144N) Not determined (ND). 

The invention claimed is:
 1. A growth hormone compound comprising one or more additional disulfide bond(s) between a loop segment and a helical segment, or within a loop segment, or between loop segments, or between helical segments and at least one additional point mutation compared to human growth hormone (hGH) as defined by SEQ ID NO: 1, wherein said additional point mutation is present in a position selected from group consisting of 40, 41, 42, 55, 57, 62, 101, 134, 136, 139, 142 and 144 of SEQ ID NO:
 1. 2. The growth hormone compound according to claim 1, wherein the growth hormone compound comprises one or more additional disulfide bond(s) connecting a loop segment and a helical segment.
 3. The growth hormone compound according to claim 1, wherein the compound comprises at least one pair of mutations selected from the group consisting of the mutations corresponding to R16C/L117C, A17C/E174C, H21C/M170C, D26C/V102C, D26C/Y103C, N47C/T50C, Q49C/G161C, F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, P61C/E66C, P61C/T67C, S71C/S132C, L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C and V185C/S188C in SEQ ID NO:
 1. 4. The growth hormone compound according to claim 3, wherein the compound comprises at least one pair of mutations selected from the group consisting of the mutations corresponding to H21C/M170C, D26C/V102C, D26C/Y103C, F54C/Y143C, F54C/S144C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C, F92C/T148C and R94C/D107C in SEQ ID NO:
 1. 5. The growth hormone compound according to claim 1, wherein the growth hormone compound comprises one or more additional disulfide bond(s) wherein at least one of the cysteines thereof is present in loop 3 (L3) corresponding to AA 128-154 in SEQ ID NO:
 1. 6. The growth hormone compound according to claim 5 , wherein the growth hormone compound comprises one or more additional disulfide bonds connecting L3 with helix 2(H2) or loop 1 (L1).
 7. The growth hormone compound according to claim 1, wherein the growth hormone compound has increased protease stability compared to human growth hormone (hGH) as defined by SEQ ID NO:
 1. 8. The growth hormone compound according to claim 1, wherein at least one additional point mutation is present in L1 (corresponding to AA 36-71 of SEQ ID NO: 1) and/or L3 (corresponding to AA 128-154 of SEQ ID NO: 1).
 9. The growth hormone compound according to claim 1 wherein at least one additional point mutation is present in the middle region of L1 (corresponding to AA 40-65 of SEQ ID NO: 1) and/or the middle region of L3(corresponding to AA 135-148 of SEQ ID NO: 1).
 10. The growth hormone compound according to claim 1 comprising exactly one additional disulfide bond and at least one additional point mutation compared to human growth hormone as defined in SEQ ID NO:
 1. 11. A pharmaceutical composition comprising growth hormone compound according to claim 1 and a pharmaceutically acceptable carrier.
 12. A method of treating a disease or a state in a patient, wherein said patient benefits from an increase in the amount of circulating growth hormone compound, said method comprising administering to said patient an effective amount of the growth hormone compound according to claim
 1. 13. A method for preparing a growth hormone compound with increased stability towards proteolytic degradation, said method comprising introducing one or more additional disulfide bonds and at least one additional point mutation, compared to human growth hormone (hGH) as defined in SEQ ID NO: 1, in said growth hormone compound. 